Project summary/abstract Herpes simplex virus (HSV) establishes a life-long latent infection in neurons. Periodically, HSV reactivates from latency, which can lead to productive replication at the body surface, often resulting in clinical symptoms. Diseases resulting from HSV reactivation includes cold sores, genital lesions and ocular disease, which can manifest as keratitis or endotheliitis. Transmission to the central nervous system following reactivation can result in herpes simplex encephalitis (HSE). Without treatment, HSE has a fatality rate of 70%, and even with treatment, many survivors exhibit long-term sequelae. There is evidence that HSV latency is heterogenous in terms of the association of viral genomes with different subnuclear structures, which may result in different abilities of latent genomes to reactivate. In some neurons, HSV genomes associate with subnuclear, phase- separated structures known as PML-NBs. We were interested to determine the contribution of PML-NBs to HSV latency and reactivation. Surprisingly, we found that PML-NBs are not present in either sensory or sympathetic primary murine neurons but do form upon treatment with interferon (IFN), in particular type I IFN. HSV can establish latency in the absence of IFN. However, IFN treatment at the time of infection results in a more restricted form of latency that is less capable of undergoing reactivation, which is reversed following PML depletion. These data imply that IFN exposure during the initial window of HSV infection can have a long-term effect on the reactivation potential of the virus in a PML-dependent manner. We will test this using in vitro and in vivo models of latency in which PML is conditionally knocked-out in neurons. Viral genomes associated with PML-NBs have been found to express lower levels of the HSV latency-associated transcript (LAT). Therefore, we will also examine the effects of PML knock-out on LAT expression and investigate the direct versus indirect effects of PML using LAT deletion viruses. To determine why viral genomes associated with PML-NBs are more restricted for reactivation we will investigate the role of PML-NBs in the association of viral genomes with core histones and different repressive histone post-translational modifications and heterochromatin-associated proteins. We will also perform dSTORM super-resolution microscopy on latent viral genomes to determine the physical structure of viral genomes found in proximity to phase-separated PML-NBs. Our long-term goals are to understand how different forms of HSV latency arise and ultimately how to modify the latent genome into its most repressive form so that it is refractory to reactivation.